Optogenetic Analysis of Behavior in the Mosquito Aedes aegypti

The mosquito Aedes aegypti is an important vector of viral pathogens and serves as a model for other vector species. Pathogens are transmitted when a mosquito bites a host animal, but the neural circuits that control seeking and biting behavior remain unknown. This paper details methods for manipulating neural activity in the mosquito using optogenetics, a key technique for determining the causal relationship between neural circuits and behavior. These methods include rearing mosquitoes for optogenetics and three assays designed to measure different steps in the sequence of arousal, attraction, proboscis probing, and engorgement on host blood. These behaviors occur at different spatial scales and in response to different sensory stimuli. Each behavioral assay is outfitted with red (~625 nm) LEDs for optogenetic activation. To detect arousal in response to olfactory stimuli, flight and walking are quantified. To assay attraction or landing, mosquitoes are presented with a heated blood meal in a large arena. Proboscis probing and engorgement are assayed with higher resolution video, enabling measurement of appendages and abdominal size. The protocol describes machine vision models to enable high-resolution temporal quantification of behavior and endpoint measurements of feeding. These methods can be used to test the role of any genetically accessible population of neurons in mosquito biting behavior and can be extended to additional behaviors.